人细胞色素P450 4A11基因核心启动子区的鉴定

The cloning and characterization of CYP4A11 gene promoter region

目的研究细胞色素P450 4A11基因启动子区对该基因转录活性的影响。方法运用生物信息学的方法预测细胞色素P450 4A11基因启动子区的重要调控序列,然后采用5’侧翼区缺失的方法将包含重要调控序列的启动子克隆到双报告基因载体中,用脂质体转染HepG2细胞,24h后检测荧光素酶相对活性。然后使用Alibaba2.0 Transfac4.0预测增强子区域可能存在的转录因子结合位点。结果·36～-720～+1、-972～+1、-1039～+1区域的启动子活性较高,其中-972～+1区域最强,与此相比-536～+1、-720～+1荧光素酶相对活性分别下降了96.9%和70.1%。结论人CYP4A11基因5’侧翼区的影响转录活性的启动子关键区域位于-972～-720bp范围内。

Objective Cytochrome P450 4A11 （ CYP4A11 ） may play an important role in the regulation of blood pressure via its metabolites 19/20-Hydroxyeicosatetraenoic acids （20-HETE） and the present study identified the promoter sequence and 5＇ -flanking region of CYP4A11 gene and investigated the molecular mechanisms through which regulates the expression of CYP4A11 gene . Method To functional analysis of CYP4A11 promoter in HepG2 ceils, Luciferase reporter plasmids containing vector alone （ pGL3-Basic ） and serially truncated putative CYP4A11 promoter fragments （1641bp-536 kb ） which were clone based on the prediction of onlinesoftware of UCSC , were transiently transfected into HepG2 ceils. Furthermore, we investigated potential transcription factor binding sites in the region fuctions as an ellhancer using the prediction programs Transfac4.0. Results The highest level of CYP4A11 promoter activity was exhibited by the - 972bp promoter construct. Reducing the promoter length by 252 bp （-720bp promoter construct） and further 184 bp （-536bp promoter construct） caused a significant decrease in activity, by 70. 1% and 96.9% in HepG2 ceils, respectively （P 〈 0.001 ）. Conclution These data indicate that positive regulatory elements may locate in the regions from -972 to -536bp of the CYP4A11 gene.